Drotrecogin alfa (activated) does not impair platelet receptor expression in vitro

Introduction

During the past decade attempts were made to find a drug decreasing the high mortality in severe sepsis and septic shock. Recently, a trial showed a significant reduction in mortality under the application of drotrecogin alfa (activated) (Xigris; Eli Lilly, Bad Homburg, Germany) (recombinant human activated protein C [APC]). Severe thrombocytopenia might be a risk factor for serious bleeding under this therapy. The median plasma concentration of APC at steady state is 0.045 μg/ml. A previous study revealed an APC-dependent reduction of CD62P on the platelet surface exclusively after activation with recombinant tissue factor. However, the amounts of APC used were high, above steady-state concentrations (0.5–20 μg/ml), and the effect of APC in therapeutic concentrations on the expression of platelet receptors has not yet been studied systematically.

Objective

To evaluate the influence of drotrecogin alfa (activated) on in vitro expression of platelet receptors at a therapeutic concentration.

Methods

Citrated blood was drawn from healthy blood donors (n = 22, 45.5% male, age 38 ± 13 years [mean ± SD]). Exclusion criteria were smoking, diabetes and use of drugs interfering with platelet function. Blood samples were adjusted with APC to final concentrations of 0.045 μg/ml APC (group 1, therapeutic dose) and 0.225 μg/ml APC (group 2, fivefold therapeutic dose), respectively. The control group received no additional APC. To evaluate platelet reactivity, samples were activated with 5 μM thrombin-receptor-agonist-peptide-6 (TRAP-6; Bachem, Heidelberg, Germany) or 2.5 μM adenosine-di-phosphate (ADP; Sigma, Taufkirchen, Germany). Samples were incubated for 10 min at 37°C with fluorescence-labeled monoclonal antibodies (mAB) against CD62P (fluorescein isothiocyanate, FITC), CD41 (phycoerythrin [PE]), CD42b(PE), CD45(FITC) (all from Beckman-Coulter, Krefeld, Germany) or PAC-1(FITC) (Becton Dickinson, San Jose, CA, USA). Analyses were performed in a flow-cytometer (Epics XL; Beckman-Coulter). The mean fluorescence intensity was calculated for all mAB except CD45. After gating granulocytes, the percentage of CD45/41-positive complexes was counted. Both calculations were run using the WinMDI software. Statistics for intergroup differences were performed by one-way ANOVA.

Results

APC in group 1 had no significant influence on platelet activation, with and without stimulation. In group 2, CD62P and CD45/41 showed a slight but nonsignificant decrease.

Conclusion

This study demonstrates that therapeutic plasma concentrations of drotrecogin alfa (activated) have neither an influence on expression of platelet activation markers nor on platelet–granulocyte complexes in vitro. Thus, a disturbance of primary hemostasis seems unlikely.